WSP-1

WSP-1 Usage Instructions:

Preparation of Stock Solution: Remove the product from the refrigerator and allow it to equilibrate at room temperature. Centrifuge at low speed for 3-5 minutes. Add an appropriate amount of anhydrous DMSO (Dimethyl Sulfoxide) to the vial and dissolve thoroughly to prepare a 5mM stock solution. Divide the solution into single-use aliquots and store them in the freezer, avoiding repeated freeze-thaw cycles. The stock solution should remain stable for at least three months.

For specific working concentrations, please refer to the literature or adjust as needed based on your experimental requirements.

Protocol for for Fluorescent Detection of Intracellular H₂S (Fluorescence Microscopy Examination) by WSP-1 ^[1]:

1. 1.Culture HASMCs until they reach approximately 90% confluence. 24 hours before the experiment, seed the cells on 8-well culture slides at a density of 30,000 cells per well and incubate for 24 hours.
2. 2.After 24 hours, allow the cells to adhere, replace the culture medium with 400 μL of WSP-1（100 μM）working solution, and incubate at 37℃ for 30 minutes.
3. 3.Replace the WSP-1 working solution with 380 μL of standard buffer and add 20 μL of the required concentration of H₂S donor compound.
4. 4.When WSP-1 reacts with hydrogen sulfide, a detectable fluorescence signal is released at Ex/Em = 465/515 nm. After incubating for 1 hour, wash the cells with 400 μL of DPBS.
5. 5.Remove the DPBS, and at room temperature, add 200 μL of Bouin's solution to each well to fix the cells for 10 minutes. Remove excess Bouin's solution and wash the cells twice with 400 μL of DPBS.
6. 6.Use a fluorescence microscope to examine and capture the fluorescence signals.

This protocol only provides a guideline, and should be modified according to your specific needs.

Protocol for Visualizing Intracellular H₂S Using WSP-1 ^[2]:

1. 1.The roots of seedlings after treatments were transferred to 20 mM Hepes-NaOH (pH 7.5) buffer solution containing 15 μM of WSP-1.
2. 2.After being incubated in darkness at 25℃ for 40 min, the roots were washed with distilled water three times and were visualized immediately by a fluorescence microscope with a 465/515 nm and an excitation/emission filter set.
3. 3.For the detection of WSP-1 fluorescence in different reactive sulfur species, WSP-1 with final concentration of 15 μM were added into the following solutions, SDS (sodium dodecyl sulfate, 2 mM), NaHSO₄ (2 mM), NaHSO₃ (2 mM), Na₂SO₄ (2 mM), Na₂SO₃ (2 mM), Na₂S₂O₄ (2 mM), GSSG (glutathione disulfide, 2 mM), sulfonamide (2 mM), GSNO (2 mM), and NaHS (2 mM), respectively. 20 μL of the above solutions were transferred to a glass slide for the visualization with a fluorescence microscope with a 465/515 nm and an excitation/emission filter set.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1]. Martelli A, Citi V,et,al. Vascular Effects of H2S-Donors: Fluorimetric Detection of H2S Generation and Ion Channel Activation in Human Aortic Smooth Muscle Cells. Methods Mol Biol. 2019;2007:79-87. doi: 10.1007/978-1-4939-9528-8_6. PMID: 31148107.

[2]. Li YJ, Chen J, et,al. In site bioimaging of hydrogen sulfide uncovers its pivotal role in regulating nitric oxide-induced lateral root formation. PLoS One. 2014 Feb 27;9(2):e90340. PMID: 24587333